Diurnal Changes in Asparaginase Activity in Pea Leaves: II. REGULATION OF ACTIVITY

Pea leaf asparaginase is stabilized by asparagine and aspartateduring incubation. In crude extracts this effect was enhancedby products of the light reaction (NADPH, NADH, or reduced ferredoxin),but these compounds were ineffective on the purified enzyme,or in the absence of asparagine. MgATP, MgADP and oxidized ferredoxinreduced asparaginase activity in purified preparation reducedor oxidized glutathione had no effect. Asparaginase activitydoes not appear to be modulated via phosphorylation/dephosphorylation.The presence of calcium during extraction increased asparaginaseactivity more than 2-fold, but addition of calcium to extractsprepared in its absence had no effect; calmodulin had no effecton activity. Co-extraction of light- and dark-treated tissueshowed that soluble factors are not responsible for the diurnalvariation in asparaginase activity. Association of asparaginasewith membranes did not account for changes in extractable activity.Use of the protein synthesis inhibitors cycloheximide, puromycin,emetine, actinomycin D and cordycepin and the thiol proteaseinhibitor leupeptin suggested that mRNA and protein synthesisare required for the increase of asparaginase activity duringthe light period and that proteolytic degradation accounts forthe decrease during the dark. Key words: Pisum sativum, asparaginase, protein synthesis, proteolysis.